Dissecting virulence of E. coli O157:H7 using genome alignments.
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Transcript of Dissecting virulence of E. coli O157:H7 using genome alignments.
![Page 1: Dissecting virulence of E. coli O157:H7 using genome alignments.](https://reader036.fdocuments.us/reader036/viewer/2022081506/56649da05503460f94a8b9ba/html5/thumbnails/1.jpg)
Dissecting virulence of E. coli O157:H7 using genome alignments
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E. coli
• Escherichia coli is a normal resident of the mammalian gut – Most strains are non-pathogenic
– E. coli K12 – lab strain
• Some strains are pathogenic to human: E. coli O157:H7
– Cause diarrhea, cramps
– The most virulent strains of O157:H7 can result in death
• Disease symptoms caused in part by the bacterial production of Shiga toxin– Shiga toxins act to inhibit protein synthesis in the host
– Cytotoxic
• Pathogenic strains transmitted through consumption of contaminated foods, (undercooked ground meat products/vegetables watered with contaminated water)
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Pathogenesis
• Microbes living in or on a host AND• Producing negative consequences to host
– Killing host cells– Inducing an immune response in host (toxic shock)– Reducing the “fitness” of the host
• Virulence factors are proteins that facilitate the microbes ability to exploit the host
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Virulence factors
• Virulence = severity of damage to the host– Things produced by the bacterial that make the host sicker– Example: Shiga toxin in enterohemorraghic E. coli
• Why would a pathogen “want” to harm the host?– Virulence is sometime a byproduct of the bacterial
efforts to live and propagate in a host• Proteins that adhere to host cells• Proteins injected into the host to manipulate the host cell• Stx encodes a protein that facilitates attachment to the host
mucosa, but it also inhibits protein synthesis in the host cell
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Pathogenic bacteria and virulence factors
-Previously we showed a comparison of genomes of good E. coli (K-12) and bad E. coli (O157:H7)
-The genome of E. coli O157:H7 contains >1,000 extra genes in comparison to good E. coli (K-12)
-Many of these extra genes enable this strain of E. coli to cause disease in humans.
-A survey of all of the published literature has identified 394 genes that are believed to be involved in the ability of E. coli O157:H7 to cause disease, and are called virulence factors.
-These genes compose >7.5% of the genome of E. coli O157:H7 strains
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14 categories of virulence factors for E. coli O157:H7
-Adhesion
-Antigen
-Clinical isolate (up-regulated gene)
-Effacing
-Effector
-Hemolysin
-Host barriers
-Iron acquisition
-Genomic island
-Regulator
-Secretion
-Toxins
-Epithelial (up-regulated gene)
-Norepinephrine (up-regulated gene)
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adhesion23%
antigen3%
effector14%
iron acquisition3%
pathogen island4%
toxins1%
upregulated with norepinephrine
9%
hemolysin2%
defense from host barriers8%
secretion systems14%
upregulated with epithelial cells
12%
upregulated in human
clinical isolates5%
regulator1%
effacing1%
Known or putative virulence factors for E. coli O157:H7 separated into categories (n=394 genes)
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Many pathogenic strains of E. coli O157:H7
• Differ in degree of virulence
• Most virulence factors are not yet known
• How can we begin to make hypotheses about which genes
play roles in the virulence of different strains?
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-Determination of the complete E. coli sequence required almost 6 years.
-E. coli K-12 is a normal inhabitant of our intestinal tract. The presence of these good bacteria helps to prevent bad bacteria from colonizing there.
-E. coli is the preferred model in biochemical genetics, molecular biology, and biotechnology and its genomic characterization will undoubtedly further research toward a more complete understanding of this important experimental, medical, and industrial organism.
(Blattner et al. Science 1997)
The first E. coli genome sequenced: the non-pathogenic E. coli K-12 genome strain MG1655
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(Perna et al. Nature 2001)
-In 1982, Escherichia coli O157:H7 recognized as a pathogen for human disease
-Also known as EDL933 from the 1982 Michigan outbreak from ground beef
-Shiga toxin producing (STEC)
Epidemiological statistics
-47 sickened individuals, no deaths
The first pathogenic E. coli genome sequence: enterohaemorrhagic (EHEC) Escherichia coli O157:H7
strain 933 EDL
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E. coli K-12:
4,140 genes
4,639,675 bp
E. coli O157:H7
5,142 genes
5,528,423 bp
~1,000 extra genes in the genome of E. coli
O157:H7
Where are the differences?
Comparison of E. coli K-12 vs. E. coli O157:H7
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EDL933 islands
Backbone
-Areas in red are only found in E. coli K12
-Areas in orange are E. coli O157:H7-specific
-Areas in blue are conserved in both
-Regions that are unique to one genome are called “islands”
Genome Comparison of E. coli O157:H7 EDL933 (pathogenic) vs. E. coli K-12 MG1655 (non-pathogenic)
K-12 islands
(unpublished image provided courtesy of Nicole T. Perna)
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Pathogenicity islands
• Comparison of the genome structire of pathogenic vs. non-pathogenic strains indicate that pathogenesis is associated with multi-gene “islands”
• The functions of some of the genes could be inferred using BLAST– genes encoding type III secretion system,
adhesins, and phage- and plasmid-encoded genes
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(Hayashi et al. DNA Res 2001)
-In July 1996, an outbreak of E. coli O157:H7 infection occurred among schoolchildren in Sakai City, Osaka, Japan.
Epidemiological statistics
-8,938 schoolchildren sickened, 3 deaths
- We are starting to ask: What genomic differences determine differences in virulence, epidemiology, and fatality?
The completion of the 2nd E. coli O157:H7 (EHEC) genome sequence (strain Sakai)
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2006 E. coli O157:H7 outbreak from bagged spinach
-multi-state outbreak
Epidemiological Statistics (from CDC)
205 people sickened, 3 deaths
-Produce-associated outbreak strains caused higher incidence of hemolytic-uremic syndrome (HUS)
(Manning et al. PNAS 2008)
-Genome alignments can be used to find variations
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Background information regarding epidemiology of E. coli O157:H7 outbreaks
(Figures addressing these topics can be found in the work of Rangel et al. 2005)
-E. coli O157:H7 outbreaks by year 1982-2002
-Median size of E. coli O157:H7 outbreaks by year
-Transmission routes of E. coli O157:H7 outbreaks by year
-Vehicles of foodborne E. coli O157:H7 outbreaks by year
-Hemolytic uremic syndrome (HUS) and case-fatality rate per 100 outbreak-related illnesses
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Do genomes change over time?
-Yes, a genome sequence represents a “snapshot” at the time the strain was sequenced. Genomes may vary ~15% or more over time
-For example, E. coli O157:H7 strain EDL933 originally had two copies of the tellurite resistance- and adherence-conferring island (TAI) pathogenicity island.
-In the lab, it has lost one of the TAI pathogenicity islands (Nicole T Perna unpublished data)
-Do the other two genomes of E. coli O157:H7 have one or both of these islands?
-Comparative genomics allow us to start understanding differences that have occurred over time and geographical differences, and to investigate variations in the genomes that may have implications for virulence differences
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As of 2010, there are 15 E. coli O157:H7 genomes sequenced. We will have you focus on three that are all in the ASAP database
(http://asap.ahabs.wisc.edu/asap/home.php)
The three strains you will focus on are:
Escherichia coli EDL933 (EHEC) [ground beef 1982 outbreak]
Escherichia coli Sakai (EHEC) [radish sprouts 1996 outbreak]
Escherichia coli EC4042 (EHEC) [fresh bagged spinach 2006 outbreak]
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Comparative genomics using the tool Mauve: Multiple Genome Aligner
• Able to identify conserved regions of multiple genomes even in the presence of rearrangements
(Darling et al. 2004)
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Student individual projects
Investigate a known or putative virulence factor to determine if it is conserved, and if it occurs in the same place in the genomes of the three E. coli O157:H7 strains.
Also using the Mauve alignment, look for islands unique to 1 out of 3, and 2 out of the 3 E. coli O157:H7 strains
-Things to consider when analyzing genome comparisons:
#1) Are there virulence factors that have been lost or gained in the different genomic islands?
#2) Could these genes present in these genomic islands play a role in one of the virulence mechanisms?
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Here is a list of a few virulence factors
ABH-0027064 stx1B
ABH-0027065 stx1A
ABH-0025275 stx2A
ABH-0025276 stx2B
ABH-0028752 eae
ABH-0028754 tir
ABH-0024965 ureD
ABH-0026806 tccP
ABH-0025423 iha
ABH-0024997 terC
ABH-0028614 lpfC
ABH-0026805 espJ
For more putative and known virulence factors (394 identified) see attached excel spreadsheet called:
Supplemental data 1 Virulence factor list for E coli O157:H7 strain EDL933